Boeneman Kelly, Mei Bing C, Dennis Allison M, Bao Gang, Deschamps Jeffrey R, Mattoussi Hedi, Medintz Igor L
Center for Bio/Molecular Science & Engineering, U.S. Naval Research Laboratory, Washington, DC 20375, USA.
J Am Chem Soc. 2009 Mar 25;131(11):3828-9. doi: 10.1021/ja809721j.
We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically self-assembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescence resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence, altering the energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.
我们展示了一种能够特异性监测半胱天冬酶3蛋白水解活性的混合荧光蛋白半导体量子点（QD）传感器的应用。经过工程改造以表达N端半胱天冬酶3切割位点的mCherry单体红色荧光蛋白，通过金属亲和配位以比例方式自组装到量子点表面。荧光蛋白与量子点的接近使其能够作为高效的荧光共振能量转移受体发挥作用。向量子点-mCherry缀合物中添加半胱天冬酶3酶会特异性切割工程化的mCherry连接子序列，改变与量子点的能量转移，并允许对蛋白水解活性进行定量监测。这种传感方法的固有优点包括以荧光附加形式在细菌中表达蛋白酶底物、易于自组装到量子点以及能够重组修饰底物以靶向其他感兴趣的蛋白酶。
